Fluorescent proteins are used for working on different analytical problems. Besides the so-called “green fluorescent protein” (GFP) and its derivatives and other fluorescent proteins, the fluorescence of which is based on the intrinsic formation of a chromophore by chemical modification of amino acids present in the primary structure, fluorescent proteins based on modified blue light receptor proteins have been described (Drepper, T., Eggert, T., Circolone, F., Heck, A., Krauβ, U., Guterl, J.-K., Wendorif, M., Losi, A., Gartner, W., Jaeger, K.-E. (2007). Reporter proteins for in vivo fluorescence without oxygen. Nature Biotech. 25, 443-445). In these proteins, which are described in German Patent Application DE 10 2005 048 828 A1, due to a point mutation the photocycle of the cofactor flavin mononucleotide (FMN) present in these proteins is interrupted, and the absorbed energy of the incident light is emitted by fluorescence. The sequence parts responsible for fluorescence in fluorescent proteins are referred to as LOV domains (light, oxygen, voltage domains). This form of fluorescent proteins is sold by the company evocatal GmbH under the product name EVOGLOW®. In the EVOGLOW® product series a distinction is made between fluorescent proteins obtained from different organisms. For example, fluorescent proteins obtained from Bacillus subtilis are designated EVOGLOW®-Bs, and fluorescent proteins obtained from Pseudomonas putida are designated EVOGLOW®-Pp. The sequences for three of these fluorescent proteins, namely EVOGLOW® Bs1, Bs2 and Pp1 are designated as sequence numbers 1, 2 and 4, respectively, in the sequence listing.
Fluorescent proteins can be used as so-called reporter proteins in cells or organisms to observe biochemical processes. Application areas are for example the investigation of gene-regulatory mechanisms or the monitoring of biotechnological processes. For application of the fluorescent proteins in extremophilic organisms, the reporter proteins have to be adapted with regard to stability against denaturing at harsh temperature and pH conditions in order to be able to detect a fluorescence signal. Furthermore, in certain investigations such as for example in colocalization studies of proteins it is necessary to be able to visualize two different populations of reporter proteins in the same cell. This is primarily achieved by different excitation and/or emission properties (colors) of the jointly used fluorescent proteins. The fluorescent proteins described in the literature thus far all have due to their identical chromophore FMN a comparable excitation and emission spectrum, as a result of which differentiation of the proteins is not possible.